Method for the detection of Neisseria gonorrhoeae

ABSTRACT

Nucleic acid probes capable of hybridizing to rRNA sequences of Neisseria gonorrhoeae and not to rRNA sequences of non-Neisseria gonorrhoeae are described along with methods utilizing such probes for the detection of Neisseria gonorrhoeae in clinical and other samples.

This is a continuation of application Ser. No. 07/356,155, filed May 24,1989, now abandoned.

FIELD OF THE INVENTION

This invention relates to detecting bacteria belonging to the genusNeisseria and more specifically provides nucleic acid probes andcompositions along with methods for their use for the specific detectionof Neisseria gonorrhoeae.

BACKGROUND OF THE INVENTION

The term "Neisseria gonorrhoeae" as used herein, refers to the bacteriaclassified as such in Bergey's Manual of Systematic Bacteriology (N. R.Krieg [ed.], 1984, pp. 288-296, Williams and Wilkins). Detection ofNeisseria gonorrhoeae is important in various medical and public healthcontexts. Neisseria gonorrhoeae is one of the leading causes of epidemicsexually transmitted disease, with approximately one million casesreported in the United States in 1983. Infection with this pathogen canresult in a wide variety of clinical manifestations, most commonlyurethritis, cervicitis and proctitis. However, infection frequentlyresults in diseases requiring hospitalization, such as endometritis,salpingitis and pelvic inflammatory disease.

Therefore, it is an aspect of the present invention to provide nucleicacid probes for use in a hybridization assay system capable of rapidlydetecting Neisseria gonorrhoeae and which is generally applicable to alltypes of clinical samples.

The scope and severity of disease caused by this organism have resultedin the development of a variety of methods for detection from clinicalsamples, however, the only methods currently recommended by the Centerfor Disease Control, the Public Health Association or the AmericanSociety for Microbiology for detection of this organism from male orfemale specimens rely primarily on culture.

Highest culture recovery of viable organisms requires immediate platingof a freshly collected specimen on an appropriate selective culturemedium (e.g. Thayer-Martin medium) and growth in a reduced oxygenatmosphere (3-10% CO₂) at 35° C. If immediate plating is not possible,then a non-nutritive transport system can be used, as long as the sampleis held for less than six hours. If the sample cannot be plated withinsix hours, then a nutritive transport system can be employed, containinggrowth media and a source of CO₂. Following plating on selective media,cultures are incubated at 34°-36° C. in a 3%-10% CO₂ environment for28-48 hours. Colonies suspected of being Neisseria then are Gram-stainedand tested for oxidase activity. Oxidase positive, Gram-negativediplococci are indicative of Neisseria spp. and must then be confirmedas Neisseria gonorrhoeae, since the culture sites may contain a varietyof non-pathogenic Neisseria. Confirmation can be carried out in avariety of ways, most commonly by carbohydrate utilization, latexagglutination or immunofluorescence tests. However, any of theseconfirmatory tests are labor intensive and time consuming, oftenrequiring additional incubation and/or growth periods.

It is another aspect of the present invention to avoid the disadvantagesassociated with traditional culturing techniques.

An enzyme immunoassay for identification of Neisseria gonorrhoeaedirectly from clinical specimens (Gonozyme™, Abbot Laboratories,Chicago, IL) has been available for several years, however, a variety ofclinical studies have indicated that this test suffers from a lack ofsensitivity and specificity. The assay also requires 3-4 hours toperform, and requires the purchase of specific signal detectionequipment.

It is yet another aspect of the present invention to avoid thedisadvantages associated with enzyme immunoassays and to employ nucleicacid probes to detect Neisseria gonorrhoeae.

It is still another aspect to provide nucleic acid probes andhybridization techniques are described which permit the specificdetection of Neisseria gonorrhoeae in clinical specimens.

As used herein, probe(s) refer to synthetic or biologically producednucleic acids (DNA or RNA) which, by design or selection, containspecific nucleotide sequences that allow them to hybridize under definedpredetermined stringencies, specifically (i.e., preferentially, seebelow--Hybridization) to target nucleic acid sequences.

Hybridization traditionally is understood as the process by which, underpredetermined reaction conditions, two partially or completelycomplementary strands of nucleic acid are allowed to come together in anantiparallel fashion to form a double-stranded nucleic acid withspecific and stable hydrogen bonds.

Totten et al. (The Journal of Infectious Diseases, 1983, 148:462-471)describe detection of Neisseria gonorrhoeae in clinical specimens byutilizing DNA probes directed against the so-called cryptic plasmidwhich commonly is associated with this bacterium. However, it is wellknown that the presence of this plasmid in Neisseria gonorrhoeaeisolates is highly regional (occurring in from 78% to 98% of isolates indifferent parts of the United States), predicting a high degree ofvariability in any assay based on the detection of its presence in testsamples.

It is yet another aspect of the present invention to remove this sourceof variability by providing probes which hybridize to nucleic acidsequences that are common to all strains of Neisseria gonorrhoeae, butwhich do not hybridize to any commensal non-Neisseria gonorrhoeae orother flora that may be present in test samples.

Lo and Yang et al. (European Patent Application 87101215.9) describe theisolation of nucleic acid probes directed against chromosomal genes ofNeisseria gonorrhoeae. These probes are purported to specificallyrecognize six strains of Neisseria gonorrhoeae, with nocross-hybridization to six strains of Neisseria meningitidis. Welcher etal. (Nucleic Acids Research, 1986, 14:10027-10044), also describe theisolation of chromosomally-targeted DNA probes. These probes arepurported to specifically recognize the strain from which they wereselected as well as six clinical isolates of Neisseria gonorrhoeae, withno cross-hybridization to 7 other Neisseria spp., E. coli or thecommensal bacterium Branhamella catarrhalis. However, testing of theaforementioned probes against a wide variety of organisms was notreported. Also, because the chromosomal target sequences are representedin low copy number in each cell, the described assays are not verysensitive and, as a consequence, their utility is significantlyrestricted in clinical applications.

It is yet another aspect of the present invention to provide nucleicacid probes which combine high specificity for Neisseria gonorrhoeaewith high sensitivity by the utilization of probes which hybridize toribosomal RNA molecules which are present in high abundance in thetarget bacteria.

Ribosomes are of profound importance to all organisms because they serveas the only known means of translating genetic information into cellularproteins, the main structural and catalytic elements of life. A clearmanifestation of this importance is the observation that all cells haveribosomes.

Ribosomes contain three distinct RNA molecules which, at least in E.coli, are referred to as 5S, 16S and 23S rRNAs. These names historicallyare related to the size of the RNA molecules, as determined by theirsedimentation rate. In actuality, however, ribosomal RNA molecules varysomewhat in size between organisms. Nonetheless, 5S, 16S, and 23S rRNAare commonly used as generic names for the homologous RNA molecules inany bacterium, and this convention will be continued herein.

While Kohne et al. (Biophysical Journal 8:1104-1118, 1968) discuss onemethod for preparing probes to rRNA sequences, they do not provide theteaching necessary to make Neisseria gonorrhoeae-specific probes.

Pace and Campbell (Journal of Bacteriology 107:543-547, 1971) discussthe homology of ribosomal ribonucleic acids from diverse bacterialspecies and a hybridization method for quantifying such homology levels.Similarly, Sogin, Sogin and Woese (Journal of Molecular Evolution1:173-184, 1972) discuss the theoretical and practical aspects of usingprimary structural characterization of different ribosomal RNA moleculesfor evaluating phylogenetic relationships. Fox, Pechman and Woese(International Journal of Systematic Bacteriology 27:44-57, 1977)discuss the comparative cataloging of 16S ribosomal RNAs as an approachto prokaryotic systematics. These references, however, fail to relievethe deficiency of Kohne's teaching with respect to Neisseria gonorrhoeaeand, in particular, do not provide Neisseria gonorrhoeae-specific probesuseful in assays for detecting Neisseria gonorrhoeae in clinicalsamples.

Hogan et al. (European patent publication WO 88/03957) describe a numberof probes which are claimed to be specific for Neisseria gonorrhoeaerRNA. However, Hogan et al. only demonstrate that their probes hybridizeto the Neisseria gonorrhoeae strain against which they are designed-aforegone, and hardly novel, conclusion. No data is given to suggest thatany of the probes will hybridize to additional N. gonorrhoeae strains.Similarly, the specificity of the probes is defined by lack ofhybridization to only single strains of a few (8) related Neisseriaspecies. No data is provided to indicate which, if any, of the probeshave the claimed specifity, i.e. that they are inclusive for all or evenmost N. gonorrhoeae.

It is another aspect of the present invention to provide nucleic acidprobes that are well characterized with respect to their hybridizationbehavior toward Neisseria and non-Neisseria bacteria.

Ribosomal RNAs are highly structured molecules. This structure dependslargely on the same types of interactions which govern probe-targetinteractions. Therefore, not all potentially useful hybridization targetsequences are equally accessible to probes under every conceivable assaycondition.

It is yet another aspect of the present invention to provide probes andprobe sets which can hybridize to rRNA target regions which can berendered accessible to probes under normal assay conditions.

The stringency of a particular set of hybridization conditions isdefined by the length and base composition of the probe/target duplex,as well as by the level and geometry of mispairing between the twonucleic acids.

Stringency may also be governed by such reaction parameters as theconcentration and type of ionic species present in the hybridizationsolution, the types and concentrations of denaturing agents present,and/or the temperature of hybridization. Generally, as hybridizationconditions become more stringent, longer probes are preferred if stablehybrids are to be formed. As a corollary, the stringency of theconditions under which a hybridization is to take place (e.g., based onthe type of assay to be performed) will dictate certain characteristicsof the preferred probes to be employed. Such relationships are wellunderstood and can be readily manipulated by those skilled in the art.

As a general matter, dependent upon probe length, such personsunderstand stringent conditions to mean approximately 35° C.-65° C. in asalt solution of approximately 0.9 molar.

SUMMARY OF THE INVENTION

In accordance with the various principles and aspects of the presentinvention, there are provided nucleic acid probes and probe setscomprising DNA or RNA sequences which hybridize, under specificconditions, to the ribosomal RNA molecules (rRNA) or rRNA genes (rDNA)of Neisseria gonorrhoeae but which do not hybridize, under the sameconditions, to the rRNA or rDNA of other related bacteria which may bepresent in test samples. Therefore the probe(s) of the present inventionprovide the basis for development of a valuable nucleic acidhybridization assay for the specific detection of N. gonorrhoeae inclinical or environmental samples.

In our experience such nucleic acid hybridization-based assays have beendiscovered to impart enhanced performance capabilities with respect tomost currently available microbiological methods for detection ofbacteria in test samples, generally including:

a) increased sensitivity; i.e., the ability to detect said fewerbacteria in a given sample;

b) potentially significant reductions in assay cost due to the use ofinexpensive reagents and reduced labor;

c) accurate identification of even biochemically unusual strains of thetarget bacteria;

d) faster results because such tests do not require the isolation of thetarget bacterium from the sample prior to testing.

It has been discovered that other advantages incurred by directing theprobes of the present invention against rRNA include the fact that therRNAs detected constitute a significant component of cellular mass.Although estimates of cellular ribosome content vary, actively growingNeisseria gonorrhoeae may contain upwards of 20,000 ribosomes per cell,and therefore 20,000 copies of each of the rRNAs. In contrast, otherpotential cellular target molecules such as genes or RNA transcriptsthereof, are less ideal since they are present in much lower abundance.

A further unexpected advantage is that the rRNAs (and the genesspecifying them) appear not to be subject to lateral transfer betweencontemporary organisms. Thus, the rRNA primary structure provides anorganism-specific molecular target, rather than a gene-specific targetas would likely be the case, for example of a plasmid-borne gene orproduct thereof which may be subject to lateral transmission betweencontemporary organisms.

Additionally, the present invention provides a number of probes andprobe sets to Neisseria gonorrhoeae rRNA target sequences which aresufficiently similar in all Neisseria gonorrhoeae strains tested thatthey can hybridize to the target region in all such Neisseriagonorrhoeae. Advantageously, these same rRNA target sequences aresufficiently different in most non-Neisseria gonorrhoeae rRNAs that,under conditions where these probes hybridizes to N. gonorrhoeae rRNAsthey do not hybridize to most non-N. gonorrhoeae rRNAs. These probecharacteristics are defined as inclusivity and exclusivity,respectively.

Other probes of the present invention are fully inclusive for N.gonorrhoeae strains, but do also hybridize to at least some non-N.gonorrhoeae target rRNAs. They are intended to be used as part of probesets which also contain one or more of the aforementioned N.gonorrhoeae-specific probes and are designed to enhance thehybridization behavior of said N. gonorrhoeae-specific probes by, forexample, acting as signal-carrying probes (detection probes) and/or byenhancing the accessability of the target site of the N.gonorrhoeae-specific probe.

The discovery that probes could be generated with the extraordinaryinclusivity and exclusivity characteristics of those of the presentinvention with respect to N. gonorrhoeae was unpredictable andunexpected.

In addition to their hybridization properties, the probes of the presentinvention also may contain certain constituents that improve theirproper or optimal functioning under particular assay conditions. Forexample, probes may be modified to improve their resistance to nucleasedegradation (e.g. by end capping), to carry detection ligands (e.g.fluorescein, 32-P, biotin, etc.), or to facilitate their capture onto asolid support (e.g., poly-deoxyadenosine "tails"). Such modificationsare elaborations on the basic probe function which is its ability tousefully discriminate between target and non-target organisms in ahybridization assay.

BRIEF DESCRIPTION OF THE TABLES

Further understanding of the principles and aspects of the presentinvention may be made by reference to the tables wherein:

Table 1--Shows a detailed alignment of the nucleotide sequences of thepreferred 16S rRNA-targeted probes of the present invention with thetarget nucleotide sequences of a number of Neisseria strains. Thecorresponding portions of the 16S rRNAs from a number of closely relatednon-Neisseria gonorrhoeae bacteria also are shown for comparison. TargetRNA sequences are written 5' to 3', probe sequences are DNA and written3' to 5'. Probes are shown along with the "core" region of variationupon which their inclusivity and exclusivity behaviors are based. Thelower case C (c) in probes 1116 and 1117 indicates a modified cytosineresidue to which a detection ligand may or may not be attached dependingon the assay format employed (see below).

Table 2--Shows a detailed alignment of the nucleotide sequences of thepreferred 23S rRNA-targeted probes of the present invention with thetarget nucleotide sequences of Neisseria gonorrhoeae. The correspondingportions of the 23S rRNAs from Escherichia coli and Neisseriameningitidis also are shown for comparison. RNA (target) sequences arewritten 5' to 3', probe sequences are DNA and written 3' to 5'. Probesare shown along with the "core" region of variation upon which theirinclusivity and exclusivity behaviors are based.

Table 3--Exemplifies the inclusivity behavior of the preferred probestoward a representative sampling of Neisseria gonorrhoeae strains in adot blot hybridization assay.

Table 4--Exemplifies the exclusivity behavior of the preferred probestoward a representative sampling of non-Neisseria gonorrhoeae strains ina dot blot hybridization assay.

DETAILED DESCRIPTION OF THE INVENTION AND BEST MODE Probe DevelopmentStrategy

The first step taken in the development of the probes of the presentinvention involved identification of regions of 16S and 23S rRNA whichpotentially could serve as target sites for Neisseriagonorrhoeae-specific nucleic acid probes. As a practical matter, it isdifficult to predict, a priori, which non-Neisseria gonorrhoeaeorganisms might be present in any test sample.

Because of the large number of such potential non-Neisseria gonorrhoeaebacteria, demonstrating exclusivity for any given probe sequence is notonly unpredictable but also extremely difficult and laborious. A morerigorous criterion was adopted which obviates the need to know whatnon-Neisseria gonorrhoeae bacteria might be present in all test samplesthat ultimately will be screened using the probes.

This entailed knowledge of the phylogenetic relationships amongNeisseria gonorrhoeae and between Neisseria gonorrhoeae and other groupsof bacteria.

Specifically, an operating but previously unproven hypothesis wasadopted that if a region(s) of rRNA nucleotide sequence could be foundthat was different in Neisseria gonorrhoeae and its closest knownevolutionary relative, N. meningitidis, then a nucleic acid probe couldbe constructed which would distinguish between the two in ahybridization assay. Based on phylogenetic principles, it then wasextrapolated that rRNA sequences of more distantly related organisms,even though their actual identity may not necessarily be known,predictably should be as or more different in a particular region ofsequence than the aforementioned close evolutionary relative ofNeisseria gonorrhoeae. However, it cannot be predicted, a priori,whether such regions exist or if they do, where within the rRNA suchregions will be located.

As the first step in identifying regions of Neisseria gonorrhoeae rRNAwhich could potentially serve as useful target sites for nucleic acidhybridization probes, relevant portions of the nucleotide sequences ofthe 16S and 23S rRNAs from Neisseria gonorrhoeae, strains ATCC19424 (thetype strain) and ATCC9793 (a common clinical isolate), Neisseriameningitidis groups B, C and D (ATCC 13090, 13102, 13113), and Neisserialactamica strain ATCC 23970 were determined.

The nucleotide sequences were determined by standard laboratoryprotocols either by cloning (Maniatis et al., 1982, Molecular Cloning; ALaboratory Manual, Cold Spring Harbor Laboratory, New York, pp 545) andsequencing (Maxam and Gilbert, 1977, Proceedings of the National Academyof Science, USA 74:560-564: Sanger et al., 1977, Proceedings of theNational Academy of Science, USA 74:5463-5467) the genes which specifythe rRNAs, and/or by direct sequencing of the rRNAs themselves usingreverse transcriptase (Lane et al., 1985, Proceedings of the NationalAcademy of Science, USA 82:6955-6959).

The determined Neisseria rRNA nucleotide sequences were compared to oneanother and to other available rRNA nucleotide sequences. Initialphylogenetic analysis of the sequence data indicated that the Neisseriabelonged to a previously described grouping of bacteria called the Betasubdivision of the Purple Bacterial division of the eubacteria (Woese,1987, Microbiological Reviews 51:221-271).

Comparison of the sequences of Neisseria gonorrhoeae and its very closerelative Neisseria meningitidis proved especially valuable. A number ofregions of 16S rRNA sequence and 23S rRNA sequence were identified whichappeared to be different in the two species of Neisseria and betweenNeisseria gonorrhoeae and non-Neisseria bacteria. The location of thoseregions within the 16S and 23S rRNA sequences which ultimately providedthe required specificity and accessability, and which are the targetsites of the probes of the present invention are shown in Table 1 and 2.Also shown are more detailed comparisons of these probe target regionsin a variety of Neisseria and non-Neisseria bacteria. The utility ofprobes based on these observed nucleotide sequence differences wasconfirmed by extensive dot blot hybridization testing as exemplified bythe data given in Tables 3 and 4. Finally, an example of the use ofmulti-probe sets in a liquid hybridization assay format is described(data shown in Table 5).

PHYSICAL DESCRIPTION OF THE PROBES

The foregoing probe selection strategy yielded a number of probes usefulfor identifying Neisseria gonorrhoeae bacteria in samples. The followingpreferred oligonucleotide probes are disclosed herein.

16S rRNA-targeted probes

Set 1, (see Table 1a):

Probe 919: 5'-cATCGGCCGCCGATATTGGCAACGGCCTTcT-3'

Probe 1116: 5'-cCGTATTACCGCAGCTGCTGGCACGTAGTTAGCCGGTGCTTATTCTcT-3'

Probe 1117: 5'-cACAAAAGTCCTTTACAACCCGAAGGCCTTCTTCAGACACGcT-3'

Set 2, (see Table 1b):

Probe 1209: 5'-GAGGATTCCGCACATGTCAAAACCAGGT-3'

Probe 1208: 5'-cGCACCTGTGTTACGGCTCCCGAAGGCACcT-3'

23S rRNA-targeted probes

Set 3, (see Table 2a):

Probe IG700: 5'-GGACATCGCGGAATCATAGCTTTATTGC-3'

Probe IG706: 5'-CCCCGCGCTTTTCGCAGGCTTACACGTC-3'

Set 4, (see Table 2b):

Probe IG705: 5'-TTCGCTTCTCTAAGCCTATGTATTCAAC-3'

Probe 1750: 5'-TAGGATACTGCACAGAATGCAGTGGGTT-3'

Probe Set 1. Probes 919, 1116, and 1117 are targeted at three adjacentregions of the 16S rRNA (Table 1). Probe 919 is targeted at the regionof Neisseria gonorrhoeae 16S rRNA corresponding approximately tonucleotide positions 453 to 480 (using the E. coli numbering system). Anumber of other versions (i.e., length variants) of probe 919 also areindicated in Table 1. Probe 1116 is targeted at positions ca. 493 to537. Probe 1117 is targeted at positions ca. 403 to 442.

As indicated in Table 1, probe 919 is "built" around the positions ofcore variation which are most useful for discriminating betweenNeisseria gonorrhoeae and its very close relative, Neisseriameningitidis. The core sequence, CguugcCaauaucGgcggcC, in the 16S rRNAof Neisseria gonorrhoeae contains 4 sequence differences with respect tothe homologous region of Neisseria meningitidis (indicated by the uppercase letters in the core sequence (Table 1). Two of these fourdifferences also are found between N. gonorrhoeae strains 9793 and 19424(i.e. the U's at positions 458 and 477). However, these differencesresult only in G:U non-canonical pairs between probe 919 and the N.gonorrhoeae (strain 19424) 16S rRNA at those positions. It has beenexperimentally determined that probe 919 hybridizes quite efficiently toN. gonorrhoeae strain 19424 in spite of these G:U pairs (Table 1).

In contrast, a probe complementary to the N. gonorrhoeae 19424 sequencethrough the 919 target region hybridizes poorly to N. gonorrhoeae 9793and also exhibits unacceptable levels of cross hybridization to othernon-Neisseria gonorrhoeae, particularly N. meningitidis.

Also shown in Table 1 are two slighly longer versions of probe 919(probes 1114 and 1115). These make use of the same core variation asprobe 919 and exhibit similar inclusivity and exclusivity behavior asprobe 919 but their extra length promotes stable probe targethybridization at higher stringencies.

Probes 1116 and 1117 do not discriminate between Neisseria gonorrhoeaeand Neisseria meningitidis--as expected given the identity of the targetsequences for these probes in these two bacterial 16S rRNAs (Table 1).Therefore, probes 1116 and 1117 would not be useful, on their own, as aNeisseria gonorrhoeae-specific probe since discrimination between thesetwo bacteria generally is considered important for most potentialapplications of an assay which would employ such probes. However, probes1116 and 1117 do have important and novel properties that make themuseful when used in conjunction with probe 919 (see below).

Probe Set 2. 16S rRNA-targeted probes 1208 and 1209 also form a set(Table 1b). Probe 1209 is targeted at the region of Neisseriagonorrhoeae 16S rRNA corresponding to nucleotide positions 983 to 1010(E. coli numbering). Probe 1208 is targeted at positions 1024 to 1051.

Of this set, probe 1209 is N. gonorrhoeae-specific, probe 1208 isdesigned as a helper/detection probe. Probe 1209 is built around thecytosine (C) and adenosine (A) differences found to exist between the N.gonorrhoeae and N. meningitidis 16S sequences shown in table 1. The corevariation upon which the specificity of probe 1209 depends includes theregion of N. gonorrhoeae sequence UuugacauguG. Probe 1208 does notdiscriminate between N. gonorrhoeae and N. meningitidis, but is usefulwhen used in conjunction with probe 1209 in a variety of dual probeassay formats.

Probe Set 3. Probe IG700 and its helper probe IG706 form another set butare targeted at the 23S rRNA of N. gonorrhoeae. Of the pair, Probe IG700is N. gonorrhoeae-specific. IG706 is designed as the helper/detectionprobe. Probe IG700 is targeted at N. gonorrhoeae 23S rRNA positions 89to 116 (using the E. coli 23S rRNA numbering) and relies on the A and Cdifferences shown in Table 2a. The core variation upon which thespecifity of probe IG700 is based includes these positions and isdefined as the N. gonorrhoeae sequence UaugauU (Table 2a).

Probe Set 4. Probe IG705 and its helper probe 1750 form the fourth probeset described in the present invention. Like the rest of the probe setdescribed above, these two are targeted at adjacent regions of the N.gonorrhoeae rRNA. Of this pair, IG705 is N. gonorrhoeae-specific, probe1750 is designed as the helper/detection probe. Probe IG705 is targetedat N. gonorrhoeae 23S rRNA positions 156 to 182 and relies on a singleposition of difference between N. gonorrhoeae and N. meningitidis(adenosine=A, Table 2b) for specifity. Probe IG705, therefore, isroughly centered about this position just as the above described N.gonorrhoeae-specific probes 919, 1209 and IG700 are centered aroundtheir respective regions of core variation. Probe 1750, the companionhelper/detection probe of this probe set is targeted at N. gonorrhoeae23S rRNA positions 126 to 154.

The specific behaviors of the probes and probe sets described above aredependent to a significant extent on the assay format in which they areemployed. Conversely, the assay format will dictate certain of theoptimal design features of particular probes. The "essence" of theprobes of the invention is not to be construed as restricted to thespecific string of nucleotides in the probes named, for example, 919 and1116 and 1117. The length of these particular oligonucleotides wasoptimized for use in the dot blot assay (and certain other anticipatedassays) described below. It is well known to one skilled in the art thatoptimal probe length will be a function of the stringency of thehybridization conditions chosen and hence the length of the instantprobes may be altered in accordance therewith. Also, in considering setscomprised of more than one probe, it is desirable that all probes behavein a compatible manner in any particular format in which they are bothemployed. Thus, the exact length of a particular probe will to a certainextent reflect its specific intended use.

The "essence" of the probes described herein resides in the discoveryand utilization of the Neisseria gonorrhoeae-specific sequencesdescribed above and given in Tables 1 and 2 (core variation).

Also note that because of the proximity of their target sites on the N.gonorrhoeae 23S rRNA, probes IG700 and IG705 (with or without the use oftheir individual helper probes) also form a useful probe set. Since bothprobes individually are specific for N. gonorrhoeae (with the notedexception of probe IG705 vs. N. flava), a dual probe assay format usingone as a capture probe and the other as a detection probe would besignificantly more "robust" than a specific/non-specificcapture/detection probe pair.

Hybridization Analysis of Probe Behavior

The sequence data in Tables 1 and 2 suggested that probes 919, 1209,IG700 and IG705 (or variations thereof) might be useful as hybridizationprobes for detecting Neisseria gonorrhoeae. However, potentially muchgreater sequence variation might exist in other Neisseria gonorrhoeaestrains not inspected by sequence analysis. Such variation might reduceor eliminate hybridization by the prospective probes to some or manyuntested Neisseria gonorrhoeae strains.

Equally as important as the inclusivity behavior of the probes, is theirexclusivity behavior, i.e., their reactivity toward non-Neisseriagonorrhoeae bacteria. The discovery of the few small stretchs ofsequence variation between the rRNAs of Neisseria gonorrhoeae andNeisseria meningitidis shown in Tables 1 and 2 was unanticipated andunexpected. However, as discussed above these patterns of sequencedifference might not hold for other strains of Neisseria meningitidis,strains of other Neisseria species or other non-Neisseria bacteria.

Therefore, the behavior of the probes toward representative Neisseriagonorrhoeae and non-Neisseria gonorrhoeae bacteria was determined byhybridization analysis.

EXAMPLE 1 Dot blot analysis of probe hybridization behavior

Dot blot analysis, in accordance with well known procedures, involvesimmobilizing a nucleic acid or a population of nucleic acids on a filtersuch as nitrocellulose, nylon, or other derivatized membrane whichreadily can be obtained commercially, specifically for this purpose.Either DNA or RNA can be easily immobilized on such a filter andsubsequently can be probed or tested for hybridization under any of avariety of conditions (i.e., stringencies) with nucleotide sequences orprobes of interest. Techniques also are available in which DNA or RNApresent in crude (unpurified) cell lysates can be immobilized withoutfirst having a purify the nucleic acid in question (e.g. Maniatis, T.,Fritsch, E. F. and Sambrook, J., 1982, Molecular Cloning:A LaboratoryManual). This latter approach was found to significantly decrease theamount of effort required to screen for particular nucleotide sequenceswhich may be present in the nucleic acids of any particular organismand, moreover, is advantageously amenable to the mass screening of largenumbers of organisms. The probes were end-labeled with radioactivephosphorous 32, using standard procedures. Following hybridization andwashing as described above, the hybridization filters were exposed toX-ray film and the intensity of the signal "scored" with respect tocontrol spots of known amount of target material (cells or RNA)visually.

Under stringent conditions, probes whose nucleotide sequences havegreater complementarity to the target sequence will exhibit a higherlevel of hybridization than probes containing less complementarity. Forthe oligonucleotide probes described herein, hybridization to rRNAtargets at 60° C. for 14-16 hours (in a hybridization solutioncontaining 0.9M NaCl, 0.12M Tris-HCl, pH 7.8, 6 mM EDTA, 0.1M KPO4, 0.1%SDS, 0.1% pyrophosphate, 0.002% ficoll, 0.02% BSA, and 0.002%polyvinylpyrrolidone), followed by three 15 minute post-hybridizationwashes at 60° C. (in 0.03M NaCl, 0.004M Tris-HCl, pH 7.8, 0.2 mM EDTA,and 0.1% SDS) to remove unbound probes, would be sufficiently stringentto produce the levels of specificity demonstrated in Tables 1 and 2.

Tables 3 and 4, illustrate representative results of dot blothybridization testing of the some 28 strains/isolates of Neisseriagonorrhoeae. A small but representative sampling of strains, isolatedfrom different clinical sources, was obtained from the American TypeCulture Collection (ATCC). The rest were isolates obtained from variousculture collections and patient populations as indicated in Tables 3 and4. Only results using the N. gonorrhoeae-specific probes of the presentinvention are shown. The various helper probes are not N.gonorrhoeae-specific as can be inferred from the sequence comparisonsshown in Tables 1 and 2.

The inclusivity behavior of probes 919, 1209, IG700, and IG705 can besummarized as follows: All probes hybridize to (i.e. are fully inclusivefor) all N. gonorrhoeae tested.

The various helper probes described above were not tested in thehybridization experiments shown in Tables 3 and 4, which were designedto test the inclusivity and exclusivity behavior of the N.gonorrhoeae-specific probes. The helper/detection probes are not N.gonorrhoeae-specific. By analysis of the available 16S and 23S rRNAsequences it can be predicted with good confidence that they willhybridize to all N. gonorrhoeae (and a good many other) bacteria. Suchhelper probes influence the kinetics and thermodynamics of thehybridization reaction, shifting the distribution of products favorablytoward formation of the desired inter-molecular probe/target complexes.

The date shown in Table 3 show that the inclusivity behavior of theprobes toward N. gonorrhoeae strains is excellent. Because the N.gonorrhoeae strains tested (Table 3) were selected as a broadrepresentation of that species, the inclusivity behavior of the probeswith respect to additional N. gonorrhoeae strains can be predicted to bequite good.

With respect to exclusivity (i.e., hybridization to non-Neisseriagonorrhoeae) the probes also behave quite similarly. With the minorexception of probe IG705 (which hybridized weakly to one tested strainof N. flava Table 4), none of the probes hybridized to any non-N.gonorrhoeae bacterium tested. As is evident in Table 4, lack ofhybridization by the probes to N. meningitidis was tested most carefully(19 strains). N. meningitidis is the closest genetic relative of N.gonorrhoeae known (by DNA hybridization studies). N. meningitidis alsois known to be found occasionally in samples which commonly are testedfor N. gonorrhoeae. For both of these reasons, the ability of the probesto discriminate between N. gonorrhoeae and N. meningitidis is deemed tobe quite important.

Except as noted above, none of the probes hybridize to any otherNeisseria species or any non-Neisseria bacterium tested.

The discovery that probes could be generated with the extraordinaryinclusivity and exclusivity characteristics of those of the presentinvention with respect to Neisseria gonorrhoeae was unpredictable andunexpected.

Description and Utility of the Probe Sets

In addition to the N. gonorrhoeae-specific probes 919, 1209, IG700 andIG705 described above, a number of probe sets including these probesplus other helper probes are described. In the simplest cases the helperprobes do no more than increase the hybridization efficiency of theaforementioned N. gonorrhoeae-specific probes by assisting those probesin gaining access to their target sequences. The following sets ofprobes having this property of improved hybridization are as follows:

    ______________________________________                                        Probe       N. gonorrhoeae-                                                                            Helper                                               Set         specific probe                                                                             Probe(s)                                             ______________________________________                                        1            919         1116, 1117                                           2           1209         1208                                                 3           IG700        IG706                                                4           IG705        1750                                                 ______________________________________                                    

In addition to structure-opening functions, such "helper" probes mayalso incorporate other functions. For example, in certain sandwich-typehybridization assay formats two probes are required to generate apositive signal. One, usually the target-organism-specific probe ismodified in such a way that it not only hybridizes to the targetmolecule but simultaneously or subsequently also can be captured out ofthe sample matrix onto a solid support surface. The other probe of theset also must hybridize to the target and, in addition, is modified tocontain a detectable ligand. For example, probes 1116 and 1117 havesignificant value as companion probes to probe 919 for use in any of avariety of dual probe, sandwich-type hybridization assay formats (e.g.the homopolymer capture, dual probe, liquid hybridization formatdescribed in U.S. Ser. Nos. 277,579, 169,646, or 233,683). In such anapplication, probe 919, or a derivative thereof would be modified at its3' terminus to contain a tract of deoxyadenosine (dA) residues ca.20-200 residues long. This would be used to "capture" the target rRNA(following liquid hybridization) from the test sample onto a solidsupport (e.g., beads, plastic surface, filter, etc.) which had beensuitably derivatized with poly-deoxythymidine (dT) for this purpose.Probes 1116 and/or 1117 (or derivatives thereof) would be used as thedetection probe and would be derivatized by some detectable ligand (e.g.32-P, fluorescein, biotin, etc.) The modified cytosine residuesindicated in Table 1 are useful as a convenient means of attaching sucha ligand. Detection of the presence of the target nucleic acid in a testsample then is indicated by the capture of the detection ligand onto thesolid surface through the series of hybridization interactions: ##STR1##

The detection probe can only become bound to the solid support if thetarget nucleic acid is present in the test sample.

The physical proximity of the target sites of the capture and detectionprobes minimizes the chance that ribonuclease present in some testsamples might negatively impact the test result by severing the targetrRNA molecule between these target sites. Probes 1116 and 1117 also aredesigned to enhance the hybridization of probe 919 by opening up thesecondary structure which involves the target region of the latter.

Likewise, the other described "helper" probes enhance the behavior ofthe N. gonorrhoeae-specific probes of their respective sets.

One further useful probe set can be defined based on the probe setsdescribed above. This is a combination of probe set 3 and 4, and isdesignated probe set 5.

    ______________________________________                                        Probe       N. gonorrhoeae-                                                                            Helper                                               Set         specific probe                                                                             Probe(s)                                             ______________________________________                                        5           IG700, IG705 IG706, 1750                                          ______________________________________                                    

In the dual probe, sandwich hybridization assay format described above,the probes of probe set 5 could be used to advantage in several ways.for example, both IG700 and IG705could be used as specific captureprobes in order to enhance the efficiency of hybridization to the N.gonorrhoeae 23S rRNA target molecules. In this case the helper probescould still be used as non-specific detection probes and also as helpersto open up the target structure. This, in principle, would increase thesignal obtainable from the assay by capturing more 23S target moleculesthan would be captured by using a single capture probe.

Another possible variation which would add "robustness" to the assaywould be to use IG700 as the capture probe and IG705 as the detectionprobe (or vice versa). Unmodified helper probes then would serveprimarily in their structure opening capacity. But in this format boththe capture and detection probes would advantageously be specific forthe target nucleic acid. This makes it less likely that a false positivehybridization signal would be generated, for example, by low levelhybridization of a non-specific detection probe to the target nucleicacid. In general, such a dual specific probe assay is preferably to aspecific/non-specific one and can often be advantageously used toincrease both hybridization efficiency and specifity of the assay.

While the description of the invention has been made with reference todetecting rRNA, it will be readily understood that the probes describedherein and probes complementary to those described herein also will beuseful for the detection of the genes (DNA) which specify the rRNA(i.e., "rDNA") and, accordingly, such probes are to be deemedequivalents to the described probes and encompassed within the spiritand scope to the present invention and the appended claims.

                                      TABLE 1                                     __________________________________________________________________________    NEISSERIA GONORRHOEAE 16S rRNA CORE AND PROBE SEQUENCE INFORMATION            __________________________________________________________________________    A) The Probe 919 Target Region.                                               Position # (E. coli) Escherichia coli                                                     ##STR2##                                                          Proteus vulgaris                                                                         CGCGUGUAUGAAGAAGGCCUUAGGGUUGUAAAGUACUUUCAGCGG                      Vibrio harveyi                                                                           CGCGUGUGUGAAGAAGGCCUUCGGGUUGUAAAGCACUUUCAGUCG                      Pseudomonas                                                                              CGCGUGCA GGAUGAAGGCCCUCGGGUUGUAAACUGCUUUUGUACG                     testosteroni                                                                  Neisseria gonorrhoeae                                                                    NGCGUGUCUGAAGAAGGCCUUCGGGUUGUAAAGGACUUUUGUCAG                      9793                                                                          Neisseria gonorrhoeae                                                                    ------------------N--------------------------                      19424                                                                         Neisseria meningitidis                                                                   C------- -------------------------------------                     13090                                                                         Neisseria meningitidis                                                                   C--------------------------------------------                      13102                                                                         Neisseria meningitidis                                                                   C------------- -----N-------------------------                     13113                                                                         Neisseria lactamica                                                                      C-------------------N---------------N------G-                      23970                                                                         Probe 1117 TcGCACAGACTTCTTCCGGAAGCCCAACATTTCCTGAAAACA c-5'                    Position # (E. coli) Escherichia coli                                                     ##STR3##                                                          Proteus vulgaris                                                                         GGAGGAAGGUGAUAAAGUUAAUACCUUUGUCAAUUGACGUUACCC                      Vibrio harveyi                                                                           UGAGGAAGGUAGUGNAGUUAAUAGCUGCAUUAUUUGACGUUAGCG                      Pseudomonas                                                                              GAACGAAAAGCCUGGGGCUAAUAUC CCCGGGUCAUGACGGUACCG                     testosteroni                                                                  Neisseria gonorrhoeae                                                                    GGAAGAAAAGGCCGUUGCCAAUAUCGGCGGCCGAUGACGGUACCU                      9793                                                                          Neisseria gonorrhoeae                                                                    ------------U------------------U-------------                      19424                                                                         Neisseria gonorrhoeae                                                                    ------------U---- ----------------------------                     8375                                                                          Neisseria meningitidis                                                                   ------------U-----U------A-----U-------------                      13090                                                                         Neisseria meningitidis                                                                   ------------U-----N------ A-----U-------------                     13113                                                                         Neisseria meningitidis                                                                   ------------NN----U------A-----U-------------                      13102                                                                         Neisseria lactamica                                                                      -----------U--GG-UU----C-CU----U------ ------C                     23970                                                                         Core variationProbe 919Probe 1114Probe 1115                                               ##STR4##                                                          Position # (E. coli) Escherichia coli                                                     ##STR5##                                                          Proteus vulgaris                                                                         GCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGGAGG                Vibrio harveyi                                                                           ACNGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGGAGN                Pseudomonas                                                                              UAAGAAUAAGCACCGGCUAACUACGUGCCAGCAGCCGCGGUAAUACGUAGG                testoseroni                                                                   Neisseria gonorrhoeae                                                                    NAAGAAUAAGCACCGGCUAACUACGUGCCAGCAGCCNCGNNNAUACGUAGG                9793                                                                          Neisseria gonorrhoeae                                                                    G--------------------------------------------------                12424                                                                         Neisseria meningitidis                                                                   G------------------ --------------------------------               13090                                                                         Neisseria meningitidis                                                                   G-----N--------------------------------------------                C13102                                                                        Neisseria meningitidis                                                                   G-----N------- -------------------------------------               13113                                                                         Neisseria lactamica                                                                      G-----------------------------------G--GU----------                23970                                                                         Probe 1116 TcTCTTATTCGTGGCCG ATTGATGCACGGTCGTCGACGCCATTATGCc-5'               B) The Probe 1209 Target Region.                                              Position # (E. coli) Escherichia coli                                                     ##STR6##                                                          Proteus vulgaris                                                                         CCUUACCUACUCUUGACAUCCAGCGAAUCCUUUAGA                               Vibrio harveyi                                                                           CCU-ACCUACUCUUGACAUCCAGGAAACUUUCCAGA                               Pseudomonas                                                                              CCUUACCCACCUUU GACAUGGCAGGAACUUACCAGA                              testosteroni                                                                  Neisseria gonorrhoeae                                                                    CCUUACCUGGUUUUGACAUGUGCGGAAUCCUCCGGA                               9793                                                                          Neisseria gonorrhoeae                                                                    --------N---------------------------                               19424                                                                         Neisseria gonorrhoeae                                                                    ---------------------------- --------                              8375                                                                          Neisseria gonorrhoeae                                                                    ------------------------------------                               2013                                                                          Neisseria meningitidis                                                                   -----------C----------A-------------                               13090                                                                         Neisseria meningitidis                                                                   --NN-------C--- -------A----------A--                              gts1349                                                                       Neisseria meningitidis                                                                   -----------C----------A-------------                               gts1346                                                                       Neisseria lactamica                                                                      -------C--------------A-------------                               23970                                                                         Core variation                                                                           Uuug acauguG                                                       Probe 1209 TGGACCAAAACTGTACACGCCTTAGGAG-5'                                    Position # (E. coli) Escherichia coli                                                     ##STR7##                                                          Proteus vulgaris                                                                         AGAGGAGUGCCUU--CGGGAACCGUGAGACAGGUGCUGCAUG                         Vibrio harveyi                                                                           GGAUUNGUGCCU---CGGGAACCGUGAGACAGGUGCUGCAUG                         Pseudomonas                                                                              GGUUUGGUGCUCGAAAGAGAACCUGCACACAGGUGCUGCAUG                         testosteroni                                                                  Neisseria gonorrhoeae                                                                    GGAGGNGUGCCUU--CGGGAGCCGUAACACAGGUGCUGCAUG                         9793                                                                          Neisseria gonorrhoeae                                                                    -----A------------------------------------                         8375                                                                          Neisseria gonorrhoeae                                                                    ----NA-- ----------------------------------                        2013                                                                          Neisseria meningitidis                                                                   ----N-N-----------------------------------                         gts1349                                                                       Neisseria meningitidis                                                                   ----N---------------- -N-------------------                        gts1346                                                                       Neisseria lactamica                                                                      --G--N------------------------------------                         23970                                                                         Probe 1208 TcCACGGAA--GCCCTCGGCATTGTGTCCACGc-5'                               __________________________________________________________________________

                                      TABLE 2                                     __________________________________________________________________________    NEISSERIA GONORRHOEAE 23S rRNA CORE AND PROBE SEQUENCE INFORMATION            __________________________________________________________________________    A) The Probe IG700 Target Region.                                             Position # (E. coli) Escherichia coli                                                         ##STR8##                                                      Neisseria gonorrhoeae 9793                                                                   CGAAGGACGUGUAAGCCUGCGAAAAGCGCCGGGGAG                           Neisseria meningitidis 13090                                                                 ----------------------------------- -                          Probe IG706    CTGCACATTCGGACGCTTTTCGCGCCCC-5'                                Position # (E. coli) Escherichia coli                                                         ##STR9##                                                      Neisseria gonorrhoeae 9793                                                                   NUGGCAAUAAAGCUAUGAUUCCGCGAUGUCCGAA                             Neisseria meningitidis 13090                                                                 C------------A-----C------------- -                            Core variation UaugauC                                                        Probe IG700    CGTTATTTCGATACTAAGGCGCTACAGG-5'                                B) The Probe IG705 Target Region.                                             Position # (E. coli) Escherichia coli                                                         ##STR10##                                                     Neisseria gonorrhoeae 9793                                                                   GGGAAACCCACUGCAUUCU-GUGCAGUAUCCUA                              Neisseria meningitidis 13090                                                                 ---------------------- -----------                             Probe 1750     TTGGGTGACGTAAGA-CACGTCATAGGAT-5'                               Position # (E. coli) Escherichia coli                                                         ##STR11##                                                     Neisseria gonorrhoeae 9793                                                                   AGUUGAAUACAUAGGCUUAGAGAAGCGAACCCG                              Neisseria meningitidis 13090                                                                 --------------A------- -----------                             Core variation G                                                              Probe IG705    CAACTTATGTATCCGAATCTCTTCGCTT-5'                                __________________________________________________________________________

                                      TABLE 3                                     __________________________________________________________________________    INCLUSIVITY DOT BLOT HYBRIDIZATION DATA                                                              PROBE                                                                         HYBRIDIZATION INDEX                                    GENUS.SPECIES                                                                            STRAIN                                                                              SOURCE                                                                              919 1209                                                                              IG700                                                                             IG705                                      __________________________________________________________________________    Neisseria gonorrhoeae                                                                    9793  (1)   +++ +++ +++ ++                                         Neisseria gonorrhoeae                                                                    27632 (1)   +++ +++ +++ ++                                         Neisseria gonorrhoeae                                                                    27633 (1)   +++ +++ +++ ++                                         Neisseria gonorrhoeae                                                                    31426 (1)   +++ +++ +++ ++                                         Neisseria gonorrhoeae                                                                    19424 (1)   +++ +++ +++ ++                                         Neisseria gonorrhoeae                                                                    27628 (1)   +++ +++ +++ ++                                         Neisseria gonorrhoeae                                                                    44    (2)   +++ +++ +++ ++                                         Neisseria gonorrhoeae                                                                    042   (2)   +++ +++ +++ ++                                          Neisseria gonorrhoeae                                                                   H02   (2)   +++ +++ +++ ++                                         Neisseria gonorrhoeae                                                                    Syv17 (2)   +++ +++ +++ ++                                         Neisseria gonorrhoeae                                                                    27629 (1)   +++ +++ +++ ++                                         Neisseria gonorrhoeae                                                                    27630 (1)   +++ +++ +++ ++                                         Neisseria gonorrhoeae                                                                    27631 (1)   +++ +++ +++ ++                                         Neisseria gonorrhoeae                                                                    10896 (1)   +++ +++ +++ ++                                         Neisseria gonorrhoeaa                                                                    GC-18 (4)   +++ +++ +++ ++                                         Neisseria gonorrhoeae                                                                    GC-17 (4)   +++ +++ +++ ++                                         Neisseria gonorrhoeae                                                                    GC-24 (4)   +++ +++ +++ ++                                         Neisseria gonorrhoeae                                                                    GC-23 (4)   +++ +++ +++ ++                                          Neisseria gonorrhoeae                                                                   GC-16 (4)   +++ +++ +++ ++                                         Neisseria gonorrhoeae                                                                    GC-20 (4)   +++ +++ +++ ++                                         Neisseria gonorrhoeae                                                                    GC-22 (4)   +++ +++ +++ ++                                         Neisseria gonorrhoeae                                                                    GC-19 (4)   +++ +++ +++ ++                                         Neisseria gonorrhoeae                                                                    G14FA171                                                                            (5)   +++ +++ +++ ++                                         Neisseria gonorrhoeae                                                                    405   (3)   +++ +++ +++ ++                                         Neisseria gonorrhoeae                                                                    410   (3)   +++ +++ +++ ++                                         Neisseria gonorrhoeae                                                                    413   (3)   +++ +++ +++ ++                                         Neisseria gonorrhoeae                                                                    444   (3)   +++ +++ +++ ++                                         Neisseria gonorrhoeae                                                                    446   (3)   +++ +++ +++ ++                                         __________________________________________________________________________     HYBRIDIZATION INDEX                                                           +++ = Positive control level of hybridization                                 ++ = Strong hybridization                                                     - = No detectable hybridization                                               SOURCE KEY:                                                                   (1) American Type Culture Collection, Rockville, MD                           (2) Clinical isolates  Dr. Michael Spence, Hahnemann University,              Philadelphia, PA                                                              (3) Clinical isolates  Dr. M. Haines, University of Miami, Miami, FL          (4) Clinical isolates  Massachusetts State Health Laboratory, Jamaica         Plain, MA                                                                     (5) Clinical isolates  Jan Cannon, University of North Carolina, Chapel       Hill, NC                                                                 

                                      TABLE 4                                     __________________________________________________________________________    EXCLUSIVITY DOT BLOT HYBRIDIZATION DATA                                       __________________________________________________________________________    A) Hybridization vs. non-gonorrhoeae Neisseria.                                                            PROBE                                                                         HYBRIDIZATION INDEX                              GENUS.SPECIES.SEROTYPE                                                                        STRAIN SOURCE                                                                              919                                                                              1209                                                                             IG700                                                                             IG705                                  __________________________________________________________________________    Neisseria meningitidis                                                                        13077  (1)   -  -  -   -                                      Neisseria meningitidis                                                                        13090  (1)   -  -  -   -                                      Neisseria meningitidis                                                                        13102  (1)   -  -  -   -                                      Neisseria meningitidis                                                                        13113  (1)   -  -  -   -                                      Neisseria meningitidis                                                                        GC-1   (4)   -  -  -   -                                      Neisseria meningitidis                                                                    A   M1 7880                                                                              (6)   -  -  -   -                                      Neisseria meningitidis                                                                    C   M2 1381                                                                              (6)   -  -  -   -                                      Neisseria meningitidis                                                                    B   1126   (3)   -  -  -   -                                      Neisseria meningitidis                                                                    B   1058   (3)   -  -  -   -                                      Neisseria meningitidis                                                                    B   14     (3)   -  -  -   -                                      Neisseria meningitidis                                                                    Non-A-D,X-Z,W-135                                                                        (3)   -  -  -   -                                      Neisseria meningitidis                                                                    B   116    (2)   -  -  -   -                                      Neisseria meningitidis                                                                    B   2341   (2)   -  -  -   -                                      Neisseria meningitidis                                                                    X   2733   (2)   -  -  -   -                                      Neisseria meningitidis                                                                    Y   140    (2)   -  -  -   -                                      Neisseria meningitidis                                                                    B   131    (2)   -  -  -   -                                      Neisseria meningitidis                                                                    SR  174    (2)   -  -  -   -                                      Neisseria meningitidis                                                                    W135                                                                              2213   (2)   -  -  -   -                                      Neisseria meningitidis                                                                    B   366    (3)   -  -  -   -                                      Neisseria cinerea                                                                             14685  (1)   -  -  -   -                                      Neisseria denitrificans                                                                       14686  (1)   -  -  -   -                                      Neisseria elongata                                                                            25925  (1)   -  -  -   -                                      Neisseria flava C11CTM1.5                                                                            (4)   -  -  -   ++                                     Neisseria flavescens                                                                          13120  (1)   -  -  -   -                                      Neisseria lactamica                                                                           23970  (1)   -  -  -   -                                      Neisseria lactamica                                                                           C1     (5)   -  -  -   -                                      Neisseria mucosa                                                                              19696  (1)   -  -  -   -                                      Neisseria polysaccharea                                                                       36088  (1)   -  -  -   -                                      Neisseria sicca 30016  (1)   -  -  -   -                                      Neisseria subflava                                                                            14799  (1)   -  -  -   -                                      __________________________________________________________________________     HYBRIDIZATION INDEX                                                           +++ = Positive control level of hybridization                                 ++ = Strong hybridization                                                     - = No detectable hybridization                                               SOURCE KEY:                                                                   (1) American Type Culture Collection, Rockville, MD                           (2) Clinical isolates  Dr. LaiKing Ng, Laboratory Centre for Disease          Control, Ottowa, Ontario, Canada                                              (3) Clinical isolates  Massachusetts State Health Laboratory, Jamaica         Plain, MA                                                                     (4) Clinical isolates  Joan Knapp, Centers for Disease Control, Atlanta,      GA                                                                            (5) Clinical isolates  Jan Cannon, University of North Carolina, Chapel       Hill, NC                                                                      (6) Clinical isolates  M. Griffis, University of San Francisco, San           Francisco, CA                                                            

    B) Hybridization vs. non-Neisseria.                                                                        PROBE                                                                         HYBRIDIZATION INDEX                              GENUS.SPECIES   STRAIN SOURCE                                                                              919                                                                              1209                                                                             IG700                                                                             IG705                                  __________________________________________________________________________    Alcaligines faecalis                                                                          8750   (1)   -  -  -   -                                      Branhamella catarrhalis                                                                       8176   (1)   -  -  -   -                                      Chromobacterium violaceum                                                                     12472  (1)   -  -  -   -                                      Eikenella corrodens                                                                           23834  (1)   -  -  -   -                                      Kingella kingae 23330  (1)   -  -  -   -                                      Kingella denitrificans                                                                        33394  (1)   -  -  -   -                                      Moraxella osloensis                                                                           19962  (1)   -  -  -   -                                      Oligella urethralis                                                                           17960  (1)   -  -  -   -                                      Pseudomonas cepacia                                                                           13945  (1)   -  -  -   -                                      Pseudomonas testosteroni                                                                      11996  (1)   -  -  -   -                                      Vitreoscilla stercoraria                                                                      VT1    (3)   -  -  -   -                                      Acinetobacter calcoaceticus                                                                   19606  (1)   -  -  -   -                                      Citrobacter freundii                                                                          8090   (1)   -  -  -   -                                      Escherichia coli                                                                              2      (2)   -  -  -   -                                      Klebsiella pneumoniae                                                                         13883  (1)   -  -  -   -                                      Morganella morganii                                                                           25830  (1)   -  -  -   -                                      Proteus mirabilis                                                                             29906  (1)   -  -  -   -                                      Pseudomonas aeruginosa                                                                        27853  (1)   -  -  -   -                                      Vibrio parahemolyticus                                                                        17802  (1)   -  -  -   -                                      Yersinia enterocolitica                                                                       9610   (1)   -  -  -   -                                      Bacillus subtilis                                                                             23059  (1)   -  -  -   -                                      Clostridium perfringens                                                                       13124  (1)   -  -  -   -                                      Lactobacillus plantarum                                                                       8014   (1)   -  -  -   -                                      Staphylococcus aureus                                                                         25923  (1)   -  -  -   -                                      Bacteroides fragilis                                                                          25285  (1)   -  -  -   -                                      Human/CaSKi                  -  -  -   -                                      Candida albicans                                                                              18804  (1)   -  -  -   -                                      __________________________________________________________________________     HYBRIDIZATION INDEX                                                           +++ = Positive control level of hybridization                                 ++ = Strong hybridization                                                     - = No detectable hybridization                                               SOURCE KEY:                                                                   (1) American Type Culture Collection, Rockville, MD                           (2) Grace Thorne, The Children's Hospital, Boston                             (3) W. R. Strohl, The Ohio State University, Columbus, OH                

What is claimed is:
 1. A method of detecting the presence of Neisseriagonorrhoeae in a sample comprising:a) contacting said sample with anucleic acid probe which preferentially hybridizes to rRNA or rDNA ofNeisseria gonorrhoeae over rRNA or rDNA of non-Neisseria bacteria,wherein said probe is complementary to a region of the Neisseriagonorrhoeae 16S rRNA selected from the group of regions consisting ofthe region 455 through 477 and the region 983 through 1010, and theregion of the Neisseria gonorrhoeae 23S rRNA selected from the group ofregions consisting of the region 89 to 116 and the region 156 to 182; b)imposing hybridization conditions on said sample and the nucleic acidprobe to allow the nucleic acid probe to hybridize to the rRNA or rDNAof Neisseria gonorrhoeae, if present in said sample, to form hybridizednucleic acid complexes, under conditions which do not allow said nucleicacid probe to form stable hybridized nucleic acid complexes withnon-Neisseria rRNA or rDNA; and c) detecting said hybridized nucleicacid complexes as an indication of the presence of said Neisseriagonorrhoeae in said sample.
 2. The method of claim 1, wherein said probedoes not hybridize under said hybridization conditions to rRNA or rDNAof Acinetobacter calcoaceticus, Bacteroides fragilis, Branhamellacatarrhalis, Citrobacter freundii, Enterobacter agglomerans, Escherichiacoli, Gardnerella vaginalis, Haemophilus ducreyii, Haemophilusinfluenzae, Klebsiella pneumoniae, Kingella kingae, Kingelladenitrificans, Lactobacillus acidophilus, Mobiluncus mulieris, Moraxellaosloensis, Morganella morganii, Neisseria cinerea, Neisseriadenitrificans, Neisseria elongata, Neisseria flavescens, Neisserialactamica, Neisseria meningitidis A, Neisseria meningitidis B, Neisseriameningitidis C, Neisseria meningitidis D, Neisseria mucosa, Neisseriasubflava, Pseudomonas aeruginosa, Salmonella arizona, Salmonellatyphimurium, Shigella flexnerii, Staphylococcus aureus, Streptococcusagalactiae and Veillonella parvula.
 3. The method of claim 1, whereinsaid nucleic acid probe consists essentially of any one of 16S rRNAprobes 919 or 1209, or 23S rRNA probes IG700 or IG705, or theircomplementary nucleotide sequences.
 4. The method of claim 1 whereinsaid contacting step comprises contacting said sample with two nucleicacid probes, one of which is probe 919 or its complementary sequence andthe other of which is selected from the group of probe 1116, probe 1117and their complementary sequences.
 5. The method of claim 1 wherein saidcontacting step comprises contacting said sample with two nucleic acidprobes, one of which is probe 1209 or its complementary sequence and theother of which is probe 1208 or its complementary sequence.
 6. Themethod of claim 1 wherein said contacting step comprises contacting saidsample with two nucleic acid probes, one of which is probe IG700 or itscomplementary sequence and the other of which is probe IG706 or itscomplementary sequence.
 7. The method of claim 1 wherein said contactingstep comprises contacting said sample with two nucleic acid probes, oneof which is probe IG705 or its complementary sequence and the other ofwhich is probe 1750 or its complementary sequence.
 8. The method ofclaim 1 wherein said contacting step comprises contacting said samplewith two nucleic acid probes, one of which is probe IG700 or itscomplementary sequence and the other of which is probe IG705 or itscomplementary sequence.